Human Adenoviruses (Ads) are large DNA viruses that cause diseases ranging from gastrointestinal and upper respiratory tract infections to eye infections. Like many large DNA viruses, Ads have evolved a specific mechanism to escape detection by the immune system. Ads encode a gene product, E3-19K, that binds to and retains MHC class I molecules in the endoplasmic reticulum of infected cells, inhibiting their egress to the cell surface and consequently suppressing antigen presentation. There is substantial evidence suggesting that the E3-19K/MHC I association plays a role in the ability of Ads to establish and maintain viral persistence. The goal of this proposal is to use molecular, structural and functional approaches to gain in-depth knowledge of the mechanism by which the E3-19K protein targets MHC class I molecules for retention in the ER and modulates cellular immune responses. We recently made a number of key observations on recombinant, soluble E3-19K interaction with MHC class I molecules. We showed that Ad serotype 2 (Ad2) E3-19K associates with immature (peptide-free) and mature (peptide-filled) MHC class I molecules. We also showed that E3-19K targets MHC class I molecules in an allele- and locus-specific manner. Moreover, we suggested that residues 89 to 93 in the conserved region of Ad2 E3-19K form a solvent-exposed loop that is critical for its immunomodulatory function. This is consistent with our preliminary data of the first x-ray crystal structure of Ad2 E3-19K bound to HLA-A2. In this proposal, we will (1) complete the high-resolution structure determination of the Ad2 E3-19K/HLA-A2 complex. We will ask to what extent our structural observations can be generalized across E3-19K proteins of different Ad serotypes and subgroups by determining the three-dimensional structures of other E3-19K/MHC I pairs. Together, these structures will be valuable for identifying features responsible for the MHC I allele- and locus-specificity of E3-19K, (2) examine the functional relationship between E3-19K and the class I antigen presentation pathway by immunoassaying for the ability of E3-19K to associate with different conformational forms of MHC class I molecules and components of the peptide-loading complex in infected cells. We will also attempt to crystallize and determine the three-dimensional structure of E3-19K bound to a peptide-free MHC class I molecule, and (3) determine if the MHC I locus-specificity of E3- 19K leads to the selective downregulation of MHC class I molecules on infected cells and if it confers protection to lysis by natural killer (NK) cells. We will also determine whether interaction involving the transmembrane and cytosolic domain of MHC class I molecules plays a role in the locus-specificity of E3-19K. These studies have the potential to reveal a novel function for E3-19K in suppressing NK-cell mediated recognition and lysis of Ad-infected cells. Overall, in dissecting the molecular basis of how E3-19K interacts with MHC class I molecules, we will provide a better understanding of how E3-19K modulates cellular immune responses. Our findings are also expected to be relevant to understand establishment and maintenance of Ad infection in healthy individuals, and disease development in immunocompromised hosts.